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United States Patent IVIICROBIAL POLYHYDROXYLATION OF STEROIDS Application December 4, 1953 Serial No. 396,315

4 Claims. (Cl. 195-51) No Drawing.

This invention relates to processes for introducing oxygen substituents into a steroid molecule and particular ly to processes for preparing oxygenated steroids by subjecting desoxy steroids to the action of oxygenating strains of microorganisms or their oxygenating enzymes.

The discovery of the remarkable therapeutic properties of cortisone, hydrocortisone and related compounds has stimulated wide interest in finding simpler and more economical methods of preparing such compounds. Heretofore these compounds were primarily produced through highly involved synthesis necessitating a considerable number of separate steps, the number required necessarily depending upon the starting material. As an example, a typical synthesis of hydrocortisone from relatively inexpensive desoxycholic acid involves approximately forty separate reactions. The most difiicult steps in the synthesis of these compounds is the introduction of oxygen substituents at various positions of the steroid molecule, particularly at the ll, 17 and 21 carbon atoms. Because of the difficulty in introducing these oxygen substituents, proper selection of the starting material and the sequence and number of operations are important factors. The selection of the starting material requires a proper balance between cost and ease of oxygenation, which necessitates extensive research to determine which are the most promising.

The oxygenation of steroids by fermentation procedures through the action of particular oxygenating strains of microorganisms or their oxygenating enzymes has been recently proposed. For example, the U. S. Patent No. 2,602,769 issued to Herbert C. Murray and Durey H. Peterson on July 8, i952, discloses the use of oxygenating fungus of the order Mucorales and particularly of the families Mucoraceae and Choanephoraceae for producing primarily ll-oxygenated steroids. This patent also discloses the preparation of compounds oxygenated at the 6 or 8 position as well as mixtures of various other oxygenated products. The U. S. Patents No. 2,649,400, issued to Murray and Peterson, No. 2,649,401, issued to William I. Haines and Donald R. Colingsworth, and No. 2,649,402, issued to Murray and Peterson, all on August 28, 1953, disclose the use of oxygenating strains of the genera Penicillium, Streptomyecs and Aspergillus for oxygcnating the ll position of steroids. Although the processes disclosed in most of these patents are utilized primarily for the production of ll-oxygenatcd steroids certain of them disclose processes for the preparation of roducts having other oxygenated positions. The aforementioned Patent No. 2,602,769 discloses the formation of products having more than one position oxygenated. The processes disclosed in the latter patent for preparing the desired multi-oxygenated products (i. e., products which have undergone oxygenation at two or more posilions of the steroid molecule), however, results in the formation of a multiplicity of other undesired oxygenated products including mixtures of stereoisomers and position isomers, rather than an appreciable amount of a single product. 'lhe yields of. the individual desired products are very low, and, because of the different types of mixtures obtained, extremely complicated extraction procedures are required to separate them from each other. It is apparent, therefore, that it is not feasible to prepare multi-oxygcnated steroids in commercial amounts by these processes.

The desirability of being able to produce a multi-oxygenated steroid in good yield, and particularly one that has the particular positions oxygenated that are necessary for forming valuable hormones, by fermentation procedures, is readily apparent. Such a method would enable the elimination of many complex steps that were heretofore found necessary in order to produce the hormonally active steroids; and, possibly more important, it would eliminate the necessity for careful selection of the starting material, thus making possible the utilization for this purpose inexpensive materials hitherto considered impractical for hormone production.

A primary object of the present invention is to produce oxygenated steroids by a process not subject to the difficulties encountered in the processes previously available. A related object is to produce multi-oxygenated steriods by a fermentation process in economically feasible yields without the formation of undesirable sideproduets. A further object is to provide, by simple and effective processes, direct oxygenation of the 11, 17 and 21 carbon atoms of steriods using fermentation procedures. Other objects and the advantages of this invention will appear hereinafter.

In accordance with the present invention, the multioxygenation of steroids is conveniently effected in a single fermentation operation by subjecting steroids to the action of two or more oxygenating strains of microorganisms or to oxygenating enzymes produced by these microorganisms which will each individually selectively oxygenate in primarily one distinct position of the steroid molecule. A feature of this process is that it provides means for oxygenating two or more positions of a steroid molecule without the formation also of a large number of unwanted oxygenated products, such as stereoisomers and position isomers which result in low yields of the desired products and requires complicated extraction procedures to separate them from the unwanted products. The ability of the process in accordance with the invention to produce desired multi-oxygenated steroids in good yield is a new and unexpected result, since it would be expected that the different microorganisms used in the same process would interfere with each other and thereby result in low yields of the desired products either through one or more organisms poisoning the other or by other interaction resulting in the disintegration of the organisms. Quite to the contrary, however, it is found that by the use of distinct microorganisms for the oxygenation of the various carbon atoms, it is possible to maintain rigid control over the oxygenation of the steroid with a resulting high yield of desired multi-oxygenatcd product. The ability to control the multi-oxygenation is very important, since this control was heretofore irnpossible by prior methods of mold-oxygenation.

In accordance with this invention, the multi-oxygena tion may take place with only one fermentation operation, thereby eliminating the necessity of separating the components after each oxygenation period as would be necessary if each oxygenation step was performed singly. This is an advantage since it greatly increases the yield over what could he obtained in separate fermentation op erations, and since in the latter case there is always a significant portion of the desired oxygenated steroid lost through the recovery procedure used for separating the desired product from the fermentation broths. In the processes in accordance with this invention, it has been found that the loss due to the recovery procedure is substantially reduced; for example, if two carbon atoms are oxygenated, the loss is reduced approximately 50% and for oxygenations of three of the carbons, the loss is reduced approximately 67%. Because of the high cost of the compounds produced, any reduction in recovery loss has a considerable economic value. This particular charucteristic of the processes of this invention is also particularly advantageous since the decrease in recovery loss results in a gain in yield of the desired product and enables the use of strains of microorganism which although slightly less effective, from the standpoint of oxygenation are more desirable for other reasons, such as nutritional requirements, stability and availability.

The method of carrying out the processes of the invention is not critical. The various strains of the microorganism may all be added at once, or the addition may be continuous or spasmodic during the fermenting period. In like manner, only one of the microorganism may be added at the start of, or continuously or spasmodically during the fermentation period, and at the end of the period or during the fermentation cycle the next microorganism may be added in any suitable manner. Also any combination of these two procedures may be used.

One of the most unusual and an unexpected feature of this invention is that the order of the introduction of the various microorganism for oxygenating the various positions of the steroid is not critical. The oxygenation of the various positions may take place in any desired order by merely arranging the order of addition of the particular microorganisms with the desired order of oxygenation. It would be expected that use of certain microorganism of the adaptive type, because of undesirable adaptation, would lead to the formation of undesirable oxygenated steroids or the presence of certain groups would inhibit oxygenation.

The particular types of microorganisms used in the process of the invention are also not important, except the use of a microorganism which is highly selective for the particular position which is to be oxygenated is required. By a microorganism that is selective is meant one that will produce a yield of at least 30% and preferably a yield of at least 50% of the desired oxygenated steroid. The yield of desired product, however, varies according to the fermentation medium employed, and it may be desirable to lower these percentages of yield somewhat to accommodate the utilization of particular media.

Table I shows a list of microorganisms which will oxygenate in the 11 position, and the particular configuation in which the ll-oxygenation takes place.

TABLE I Microorganism Position Spondyloclndlum-.... 11 Acrotht'cium nirum lla Acrothicium lunatum. lla Acrothicz'um robuatmm 11a Acrothicz'um aerenarz'um. 11c: Currularia Zlmata (NR 116 Helmmthosporlum l1 Brachysporlumur 1t Heterosporlurm. 11 Napicladlurnm ll Diplocociurm. ll Alternaria. 1t Stemphylium 1t Asperglllns 11 Aspergillua clavatus l 11a Aspergillus fischer 11o: Aapcrgillua flaws... 11a Aapergillua itrrconzcu 11a Asperm'llus luchuemts Ila Aspems'llua nidulansm 11a Asperm'llusniaer, 11a Aspergillus ochraceus (NRRL 405-260-4718) l. 11a Asperqt'llusustus l.,..1.. 11a Aspergillus wmt:' 11a Streptomyoes ll Streptomycca jradiae, 11o:

Streptomucrsmdus j 11a Microorganism Position Penicllllu1n ..a... ll Penicillium adametz 11a Pentcillium raistrickii. 11a Penicillium urticae I la Penicillium lutzum i Penicillium ezpamum.... lla Penicillium notatum Ila Penicillium lilacinum r lla Pcnicillium brez icompoctum. Ila Penicillium citrinum 11a Penicillium thamiL. 110: Penicillium jonthimllu 11oz Penicillium ninricana 11a Panicillium uzalicum 11a Gurvularta ll Curvularic falcata ll Curuularia brachya om 11 Curvularia Innate {NRRL 2434 and 2474) 118 Curvularia pallescena (N RRL 2381) 11B Rhlropus s c l r 11 Rhizupns nim'icuna (ATCG 6277).. 11a Cunnlnghamella 11 kesleena 11B Mucor v 11 Omphalla... 11 Omphulia tralucida 11B Stlgmlna l1 Stigmina plntam' 118 For the oxygenation of the ll-position, it is preferred to use an oxygenating strain of fungi of the family Dematiaceae or oxygenating enzymes produced by these microorganisms, since these organisms are selective in their oxygenation. One group that has been found particularly elfective is of the genus Spondylocladium. For the introduction of the hydroxy group at the 11-position in the p-configuration as is present in hydrocortisone, the srtain Curvularia lunata (NRRL 2434), which may also be considered a microorganisms of. the genus Spondylocladium, is the preferred organism. The use of the species Aspergillus ochraceus (NRRL 405-260-4718) of the genus Aspergillus is also highly desirable for the oxygenation since this species will produce Ila-oxygenated products in very high yield.

The use of the other microorganisms for the oxygenation at the ll-position, although suitable in the processes of the invention, are less desirable because they do not have the selective quality characteristic of the family Dematiaceae and the species A. ochraceus, since their use results in the formation of some undesirable products. A further difiiculty is that many of these other oxygenating microorganisms, such as various species of the Mucorales, are very specific in their nutritional requirements, and, therefore, the culture mediums suitable for growing such microorganisms are limited. Also, the spores of many of the Phycomycetes, including the Mucorales, are destroyed by the lyophilization procedure; therefore, great difiiculty is encountered in retaining their desirable properties since the cultures are subject to degeneration. Further, the Mucorales grow very poorly under submerged conditions, and, if any foam is allowed to collect on the surface of the fermentation liquor, the Mucorales collect in the foam and develop as a pellicle over the surface of the culture liquor. This formation of a surface pellicle is undesirable since such growth does not permit the microorganism to come in intimate contact with the steroid contained in the main body of the fermentation broth. Under these conditions, therefore, large amounts of antifoam agents are required which complicates the isolation of the desired oxygenated steroids.

In Table II a number of microorganisms that will oxygenate in the l7-position are listed. Also, other species of the genus Trichoderma will also be effective in oxygenating the 17 carbon atom.

Table III lists a number of microorganisms that will oxygenate the 21-position. Other species under the family Sphaeroidaceae and particularly those under the genus Wojnowicia will also be effective for this position.

TABLE III llilcroorganisrn Position Sphaeroidaceae Wojnowieia gm minis (N R RL 2472) Oeratopyenis Eriosporim Prosthemlum Oryptostictos Dilophospora. Uroconis be Pyrcnoehaeta" decipiens Coniothyriwn eliz' Septcria aesculi Phopsis citrz'. Septoria apii Septoria Zycopcrsici The oxygenation of the 6 position may be accomplished by certain strains of the genus Rhizopus, such as arrhiztis and RH 176. Various other positions of the steroid may be oxygenated, such as the 7, 8 and 14 carbon atoms, by other microorganisms known to the art.

A single microorganism may be used in one or more steps to oxygenate two or more positions of the steroids, but generally this type of microorganism is undesirable since whenever a microorganism is found that will oxygenate in two desired positions, its use will often result in the formation of a multiplicity of other products of varying degree of oxygenation. By the use of such a microorganism, it is diflicult to maintain control over the oxygenation.

In carrying out the process of this invention, the steroid to be oxygenated is subjected to the action of oxygenating enzymes produced by growing oxygenating strains of fungi. This is conveniently accomplished by growing the microorganisms under aerobic conditions in a suitable nutrient medium in intimate contact with the steroid to be oxygenated; the culturing growth of the microorganism being continued until the desired oxygenation has occurred. Alternatively the process may be efiected by the use of homogenized resting cells by first growing the microorganism in a suitable fermentation medium under aerobic conditions, separating the cells from the fermentation medium and adding the steroid to these resting cells and continuing the aerobic conditions for a sufficient time to effect the desired oxygenation.

The steroid can be added to the nutrient medium as a suspension in a suitable solvent, such as water; as a solution in a solvent, such as acetone, propylene glycol dimethyl formamide or dimethylacetamide; or in a finely divided form, such as a solid micronized powder. In general, it is desirable that the steroid be present in very finely divided form in order to permit maximum contact with the oxygenating culture medium and insure completion of the reaction.

The process of the present invention can be effected in both stationary and submerged cultures of microorganisms growing under aerobic conditions, although, for practical purposes, it is most conveniently carried out by growing the microorganisms under submerged conditions in a suitable aqueous fermentation medium containing the steroid.

it is essential to this invention, however, that a fermentation medium be selected which is suitable for growth of each of the microorganisms, and the conditions under which each will oxygenate effectively must be maintained during its oxygenation period. Since the microorsnmisms may be oxygenated successively as well as concu rrcntly, it enables careful control of the conditions most favorable for each oxygenating organism.

The use of a combination of microorganism in the processes of this invention makes possible effective use of the nutrient medium, in that the utilization of the medium may be complete; whereas, in other fermentation processes, the utilization of the nutrient medium is incomplete. This factor will tend to reduce operational costs of the process and thereby efiect a further economy.

Aqueous nutrient mediums suitable for growing the oxygenating strains of the microorganism must contain sources of assimilable carbon and nitrogen as well as minor amounts of inorganic salts. Any of the usual sources of assimilable carbon, such as dextrose, glucose, inverted molasses, and the like, employed in fermentation mediums, can be used in carying out the process of our invention. Similarly, complex sources of nitrogen usually employed in commercial fermentation processes such as lactalbumin digest (Edamine) and corn steep liquor; or inorganic sources of nitrogen, such as dibasic ammonium phosphate, ammonium nitrate, and the like, are satisfactory for use in the fermentation media. Minor amounts of other substances, such as nicotinamide or inorganic salts, such as suitable soluble salts of magnesium, zinc, potassium, sodium, phosphorus, and iron are usually available in complex sources of carbon and nitrogen or may be conveniently added to the fermentation medium in m nor amounts to promote maximum growth of the oxygenating microorganism.

The following are examples of suitable aqueous nutrient mediums which can be used in our process of oily genating steroids:

Medium No. I

Commercial dextrose (cerelose) 50.00 Commercial lactalbumin digest (Edamine) 20.00 Corn steep liquor 5.0 Distilled water is added to give a total volume of 1 liter of nutrient medium and the pH adjusted to 6.5 with sodium hydroxide.

Medium N0. 2

Inverted black strap molasses 100.0 Commercial lactalhumin digest (Edamine) c 200 Corn steep liquor 5.0 Distilled water is added to give a totai volume of l liter of nutrient medium and the pH adjusted to 6.5 with sodium hydroxide.

Medium No. 3

inverted black strap molasses 100.0 Corn steep liquor 5.0

Distilled water is added to give a total volume of 1 liter of nutrient medium and the pH adjusted to 6.5 with sodium hydroxide.

7 Medium N0. 4

Inverted black strap molasses 100.0 Corn steep liquor 20.0 Distilled water is added to give a total volume of 1 liter of nutrient medium and the pH adjusted to 6.5 with sodium hydroxide.

Medium N0. 5

Inverted black strap molasses 50.0 Corn steep liquor 6.3 Distilled water is added to give a total volume of 1 liter of nutrient medium and the pH adjusted to 6.5 with sodium hydroxide.

Medium No. 6

Dextrose 50.0 (NHO HPO', 7.5 KzHPO 1.0 MgSO .7H O 0.5 KCl 0.5 FSO4.7H2O ZHSO4.7H2O Distilled water is added to give a total volume of 1 liter of nutrient medium and the pH adjusted to 6.5 with sodium hydroxide.

Medium N0. 7

Cuban blackstrap inverted molasses 50.0 Corn steep 5.0 Distilled water is added to give a total volume of 1 liter of nutrient medium and pH adjusted to 5.9 with sodium hydroxide.

Medium N0. 8

Cuban blackstrap inverted molasses 100.0 Corn steep 5.0 Distilled water is added to give a total volume of 1 liter of nutrient medium and the pH adjusted to 5.8 with sodium hydroxide.

The addition of minor amounts of anti-foaming agents, although not essential, is desirable with some fermentation media. It has been found that the addition to certain fermentation mediums, of a substituted oxazaline which is a nonvolatile, amine-type. cationic surface active agent, available under the trade name Alkaterge C, is particularly effective in reducing the amount of foam, although other antifoam agents known to be useful for this purpose can also be used.

The utilization of these processes also results in many other savings in operational costs. One such saving, in particular, is the time factor involved with the resulting saving in amount of equipment necessary and the power usage. When using these processes only one fermentcr is necessary where heretofore two or more would be required. When carrying out this process. the sterilization of the fermentation medium may be accomplished in one step, thereby effecting a further economy.

The processes of this invention are particularly useful in the oxygenation of 4-pregnene-3.20-dione, a relatively cheap material to 4-pregnene-ll;3,17a.21-triol-3,20-dione by the introduction of 115, 17a and 21 hydroxy groups. Other steroids, however, may be oxygenated thereby producing hormones or intermediates in the preparation of hormones. Thus, this process is applicable in general to saturated and unsaturated cyclopentanopolyhydrophenanthrene compounds. Such cyclopentanopolyhydrophcm anthrene compounds may be unsubstituted or may contain substituents, such as keto, hydroxyl. acyloxy, halide alkyl, and the like, at various positions of the cyclopcrr tanopolyhydrophenanthrene nucleus. In addition, such compounds may have at the I'l -position a kcto side chain, a saturated or unsaturated hydrocarbon side chain, a carboxylic acid side chain, and the like. Examples of classes of such cyclopentanopolyhydrophenanthrene compounds that might be mentioned are pregnanes, pregnenes, allopregnanes, allopregnenes, androstanes, bile acids and their esters, sterols, sapogcnins, and derivatives thereof. Thus, representative steroids, such as 4-pregnene-3,20-dione; 4- pregnene-l7a ol-3,20-dione; 4-pregnene-l7a-ol-3,20-dione- 2l-acetate; desoxycorticosterone; 4-pregnene-17a-21-diol- 3,20-dione; 3-keto-cholanie acid; lithocholic acid; diosgenin; 5,6-dichlorodiosgenin; 4-pregnene-3fi-ol-20-one; 5, 6-dichloropregnane-3B-oI-ZO-one; 5,6-dichloropregnane- 21-o1-3,20-dione; 4pregnene-6'ol-3,20-dione; and the like, can be oxygenated at positions 11, 17, 21, 6 and various other positions to obtain the corresponding multi-hydroxy derivatives.

For example, an 11,17,21-desoxy pregnene can be oxygcnated in accordance with the following procedure:

Three samples of a sterile culture medium, such as those referred to above, are first each inoculated with a different microorganism that will either selectively oxygenate at the 11, 17 or 21 position, respectively, by introducing a small amount of spore suspension or vegetative growth of the organism. The inoculated nutrient medium containing the Zl-oxygenating microorganism is then incubated at a temperature of about 20 to 45 C., while being agitated in the presence of oxygen for a period of about a few hours to several days. At this point, a solution of a 11,17,2l-desoxy pregnene in a solvent such as propylene glycol is added to the fermentation medium and the agitation and aeration of the nutrient medium continued for about 5 to 30 hours, or until the oxygenation reaction is completed. During this fermentation period the second medium containing the 17-oxygenating microorganism is incubated at a temperature of about 20 to 45 C., while being agitated in the presence of oxygen for a period of a few hours to several days. This medium is then added to the fermented desoxy pregnene medium and the agitation and aeration of the medium continued for about 5 to 30 hours. The ll-oxygenating microorganism which had been been similarly incubated is then added and the fermentation continued for about 5 to 30 hours. When this oxygenation is complete, the 11,17,21-tri-hydroxy pregnene may be recovered from the fermentation broth by extraction with a suitable Water immiscible organic solvent. Suitable solvents for this purpose that might be mentioned are chloroform, methylene chloride, Z-methyl-S-cthyl pyridine, organic acid esters, aromatic hydrocarbons, ketones and amides, and the like. The solvent solution containing the oxygenated steroid can then be evaporated to yield the desired product which may be further purified by recrystallization or other procedures conventional in the art.

Alternatively, the process of this invention can be effected by contacting the oxygenating enzymes produced by the fermentation of the microorganisms with the steroid to be oxygenated. This can be accomplished by recovering the oxygenating enzymes from the fermentation broths in accordance with procedures known in the art, and intimately contacting such enzymes with a steroid in an aqueous medium in a manner similar to the above fermentation medium procedure.

In a similar manner as above, the 11,17,21-trihydroxy pregnene may be further fermented before separation from the medium with other microorganism to oxygenate other positions. As example, the above procedure may be continued with a microorganism that will oxygenate in the 6-position such as Rhizopus arr/rims and the 6,11,17,21- tetrahydroxy pregnene is then separated from the fermentation broth.

The following examples are given for purpose of illustration.

EXAMPLE 1 Four samples of approximately 50 ml. each of a culture medium having the composition of medium #1 were sterilized for 20 minutes at C. in 250 ml. flasks. Each medium was then inoculated with approximately five milliliters of vegetative growth of culture Wojnowicia graminis (NRRL 2472). The inoculated mixtures were then agitated using a rotary shaker at an agitation speed of 220 R. P. M., for approximately 72 hours while maintaining the temperature at 28 C. A sterile solution of 20 mg. of 4-pregnene-3,20-dione in ml. of propylene was added to each flask and the agitation continued at the same rate for approximately 48 hours. At this point 50 m1. of a culture growth, having initially the composition of medium #1 and containing 5 ml. of vegetative growth Curvularia lunata (NRRL 2434), was added to each flask. The culture growth was obtained by developing the microorganism for three days in the medium. The flasks containing approximately 100 ml. were agitated and individual flasks removed after 12, 24, 48 and 72 hours incubation and extracted with chloroform. The extracts were spotted on paper strips and developed with the solvent system, benzene/ formamide using the Zafiaroni technique reported in Science 111, 6 (1950). A U. V. absorbing tetrazoliurn reducing product was formed having the same rate of mobility as 4-pregnene-11,8,21-diol-3,20-dione. A band of the oxidation product was eluted from the paper and the U. V. spectrum of its sulfuric acid chromogen obtained. It was found to be identical with that of 4- pregnene-l 1 3,2l-diol-3,20-dione.

EXAMPLE 2 Six samples of approximately 50 ml. each of a culture medium having the composition of medium #1 were sterilized for 20 minutes at 120 C. in 250 ml. flasks. Each medium was then inoculated with approximately five milliliters of vegetative growth of culture Wojnowicia gramim's (NRRL 2472). The inoculated mixtures were then agitated using a rotary shaker at an agitation speed of 220 R. P. M. for approximately 48 hours, while maintaining the temperature at 28 C. A sterile solution of mg. of 4-pregnene-3,20-dione in 2.5 ml. of propylene glycol was added to each flask and the agitation continued at the same rate for approximately 48 hours. At this point 50 ml. of a culture growth, having initially the composition of medium #1 and containing 5 ml. of vegetative growth Curvularia lunata (NRRL 2434), was added to each flask. This culture growth was obtained by developing the microorganism for three days in the medium. The flasks containing approximately 100 ml. were agitated and individual flasks were removed after 6, 12, 24, 48, 72 and 96 hours incubation and extracted with chloroform. The extracts were spotted on paper strips and developed with the solvent system benzene/formamide. A U. V. absorbing-tetrazolium reducing product was found having the same mobility as 4- pregnene-l1B,2l-diol-3,20-dione.

EXAMPLE 3 Six samples of approximately 50 ml. each of a culture medium having the composition of medium #1 were sterilized for 20 minutes at 120 C. in 250 ml. flasks. Each medium was then inoculated with approximately five milliliters of vegetative growth of culture Curvularia lunata (NRRL 2434). The inoculated mixtures were then agitated using a rotary shaker at an agitation speed of 220 R. P. M. for approximately 48 hours while maintaining the temperature at 28 C. A sterile solution of 10 mg. of 4-pregnene-l7u-ol-3,20dione in 2.5 ml. of propylene glycol was added to each flask and the agitation continued at the same rate for 24 hours. At this point 50 ml. of a culture growth, having initially the composition of medium #1 and containing 5 ml. of vegetative growth Wojnowicia graminis (NRRL 2472) was added to each flask. This culture growth was obtained by developing the microorganism for three days in the medium. The flasks containing approximately 100 ml. were agitated and individual flasks were removed after 6, 12, 24, 48, 72 and 96 hours of incubation and extracted with chloroform. The extracts were spotted on paper and developed in the system chloroform/formamide:methanol. A conversion product which was U. V. absorbing and tetrazolium reducing was observed opposite 4-pregnene-l1,8,17a,2l-triol-3,20-dione. The U. V. spectrum of the product in concentrated sulfuric acid was compared and found identical to that of 4-pregnene- 115,] 7a,2 i-triol-3,20-dione.

EXAMPLE 4 Five samples of approximately 50 ml. each of a culture having the composition of medium #1 were sterilized for 20 minutes at 120 C. in 250 ml. flasks. Each medium was then inoculated with approximately five milliliters of vegetative growth of culture Wojnowicia gramznz's (NRRL 2472}. The inoculated mixtures were then agitated using a rotary shaker at an agitation speed of 0 R. P. M. for approximately 48 hours while maintaining the temperature at 28 C. A sterile solution of 10 mg. of 4 pregnene-3,20-dione in 2.5 ml. of propylene giycol was added to each flask and the agitation continued at the same rate for 48 hours. At this point 50 ml. of a culture growth, having initially the composition of medium #1 and containing 5 ml. of vegetative growth of Trichoderma viririe (NRRL 2473), was added to each flask. This culture growth was obtained by developing the microorganism for three days in the medium. The flasks containing approximately ml. were again agitated at the same rate and temperature for 72 hours. At the end of this period 50 ml. of culture growth, having initially the composition of medium #1 and containing 5 ml. of vegetative growth of Cm'vularia lunata (NRRL 2434), was added to each flask. This growth was obtained in a manner similar to the others. The flasks now containing approximately 150 ml. were agitated and individual flasks were removed after 6. 12, 24, 48 and 72 hours of incubation and extracted with chloroform. The extracts were spotted on paper and developed in the system chloroform/iormamide:methanol. A U. V. absorbing tetrazolium reducing oxidation product was observed whose R: and sulfuric acid U. V. spectrum were identical with 4-prcgncne-lU3,l7nt,2l-triol-3,20-dione.

EXAMPLE 5 Five samples of approximately 50 ml. each of a culture medium having the composition of medium #1 were sterilized for 2%) minutes at C. in 250 ml. flasks. Each medium was then inoculated with approximately five milliliters of vegetative growth of culture Aspergillus niger. The inoculated mixtures were then agitated using a rotary shaker at an agitation speed of 220 R. P. M. for approximately 48 hours while maintaining the temperature at 28 C. A sterile solution of 20 mg. of 4-pregnene-3,20-dione in /2 ml. of dimethylformamide was added to each flask and the agitation continued for approximately 48 hours. .At this point 50 ml. of a culture growth, having initially the composition of medium #1 and containing 5 ml. of vegetative growth Trichoderma album, was added to each flask. The culture growth was obtained by developing the microorganisms for 48 hours in the medium. The flasks containing approximately 100 ml. were agitated and the flasks removed after 48 hours. One of the flasks was extracted with chloroform and spotted on a paper strip. The spot was in the region of 4-pregnene-1la,17a-diol-3,20-dione. Fifty ml. of culture growth, having initially the composition of medium #1 and containing 5 ml. of vegetative growth of Hendersonia ac'icola, (NRRL 2585), was added to each of the remaining flasks. The flasks now containing approximately ml. were agitated and individual flasks removed after 24, 48, 72 and 96 hours of incubation and extracted with chloroform. The extracts were spotted on paper strips and developed with the solvent system, benzene/formamide. The spot was in the region of 4-pregneue-l1a,17e,21-triol-3,20-dione.

EXAMPLE 6 Five samples of approximately 50 ml. each of a culture medium having the composition of medium #1 were sterilized for 20 minutes at 120 C. in 250 ml. flasks. Each medium was then inoculated with approximately five milliliters of vegetative growth of a culture of Trichoderma glaucum. The inoculated mixtures were then agitated using a rotary shaker at an agitation speed of 220' R. P. M. for approximately 48 hours, while maintaining the temperature at 28 C. A sterile solution of 20 mg. of 4-pregnene-3,20-clione in /2 ml. of dimethylformamide was added to each flask and the agitation continued at the same rate for approximately 6!.) hours. At this point 50 ml. of a culture growth, having initially the composition of medium #1 and containing ml. of vegetative growth of Woinawicia graminis (NRRL 2472), were added to each flask. This culture growth was obtained by developing the microorganism for three days in the medium. The flasks containing approximately 100 ml. were agitated at the same rate for 48 hours. One of the flasks was extracted with chloroform and spotted on a paper strip. The spot was in the region of 4-pregnene-l7a,2l-diol-3,20-dio11e. Fifty ml. of culture growth, having initially the composition of medium #1 and containing 5 ml. of vegetative growth of Cunninghamella blakesleana, were added to each of the remaining flasks. This culture growth was obtained by developing the microorganism for one day in the medium. The flasks now containing approximately 150 ml. were agitated and individual flasks were removed after 12, 24, 48 and 72 hours of incubation and extracted with chloroform. The extracts were spotted on paper and developed in the system chloroform/formamide:methanol. The spots were in the region of 4-pregnene-11p.l7a,2l-triol-3,20dione.

EXAMPLE 7 The procedure in Example 6 was followed except that the culture of Wojnowicia graminis (NRRL 2472) was used initially and then the culture of T riclzoderma glaucum followed by the Cmmz'nglmmclla blakcsleana. The results were the same as in Example 6.

EXAMPLE 8 Five samples of approximately 50 ml. each of a culture having a composition of medium #1 were sterilized for 20 minutes at 120 C. in 250 ml. flasks. Each medium was then inoculated with approximately five ml. of a vegetative growth of a culture of Penicillium adametzi. The inoculated mixtures were then agitated using a rotary shaker at an agitation speed of 220 R. P. M. for approximately 48 hours while maintaining the temperature at 28 C. A sterile solution of 20 mg. of 4-pregnene-3,20- dione in /5 ml. of dimethylformamide was added to each flask and the agitation continued at the same rate for 48 hours. At this point 50 ml. of a culture growth, having initially the composition of medium #1 and containing 5 ml. of vegetative growth of T richoderma lignorum, was added to each flask. This culture growth was obtained by developing the microorganism for three days in the medium. The flasks containing approximately 100 ml. were again agitated at the same rate and temperature for 60 hours. One of the flasks was extracted with chloroform and spotted on a paper strip. The spot was in the region of 4-pregnene-11u,17q-diol-3,20-dione. Fifty ml. of culture growth, having initially the composition of medium #1 and containing 5 ml. of vegetative growth of Wojnowicia gramim's (NNRL 2472), was added to each of the remaining flasks. The flasks now containing approximately 150 ml. were again agitated and individual flasks removed after 24, 48, 72 and 96 hours of incubation and extracted with chloroform. The extracts were spotted on paper and developed in the system chloroform/formamidezmethanol. The spot was in the region of 4- pregnene-l1a,l7a,2l-triol-3,20-dione.

EXAMPLE 9 The procedure in Example 8 was followed except that the culture of Wojnowicia graminis (NRRL 2472) was used initially and then the culture of Trichoderma lignorum followed by the Penicillium adametzi. The results were the same as in Example 8.

EXAMPLE 10 The procedure in Example 8 was followed except that the culture of T richoderma lignorum was used initially and then the culture of Wojnowicia graminis (NRRL 2472) followed by the Penicillium adametzi. The result was the same as in Example 8.

EXAMPLE 11 The procedure in Example 8 was followed except that the culture of Penicillium adametzi was used initially and then the culture Wojnowicia graminis (NRRL 2472) followed by the Trichoderma Iignorum. The result was the same as in Example 8.

EXAMPLE 12 Five samples of approximately 50 ml. each of a. culture medium having the composition of medium #1 were sterilized for 20 minutes at 120 C. in 250 ml. flasks. Each medium was then inoculated with approximately five ml. of vegetative growth of culture Trichoderma viride (NRRL 2473). The inoculated mixtures were then agitated using a rotary shaker at an agitation speed of 220 R. P. M. for approximately 72 hours while maintaining the temperature at 28 C. A sterile solution of 20 mg. of 4-pregnene-3,20-dione in /2 ml. of dimethylformamide was added to each flask and the agitation continued for approximately 48 hours. At this point 50 ml. of a culture growth, having initially the composition of medium #1 and containing 5 ml. of vegetative growth of Wojnowicia graminis (NRRL 2472), was added to each flask. The culture growth was obtained by developing the microorganism for three days in the medium. The flasks containing approximately ml. were agitated at the same rate for 48 hours. One of the flasks was then extracted with chloroform and spotted on a paper strip. The spot was in the region of 4-pregnene-l7a,2ldiol-3,20-dione. Fifty ml. of a culture growth, having initially the composition of medium #1 and containing 5 ml. of vegetative growth of Omphaliatra lucida, were added to each of the remaining flasks. The culture growth was obtained by developing the microorganism for seven days in the medium. The flasks now containing approximately 150 ml. were again agitated and individual flasks were removed after 12, 24, 48 and 72 hours of incubation and extracted with chloroform. The extracts were spotted on paper and developed in the system chloroform/formamide:methanol. A tetrazolium reducing spot in the region of 4-pregnene-l1fi,17u,21-triol-3,20- dione was observed. With a portion of the residue the U. V. spectrum is concentrated H 50 was obtained and found to be similar to 4-pregnene-l1,3,17a,21-triol3,20- dione. A second portion of extract was respotted on paper and then oxidized with dichromate in glacial acetic acid. The paper was developed and revealed a spot of the same mobility as 4-pregneneF17a,21-diol-3,11,20-trione. Another portion was tested for eosenaphil response and was found active.

EXALIPLE 13 Five samples of approximately 50 ml. each of a culture medium having the composition of medium #1 was sterilized for 20 minutes at C. in 250 ml. flasks. Each medium was then inoculated with approximately five ml. of vegetative growth of a culture of Hendersonia rubi. The inoculated mixtures were then agitated using a rotary shaker at an agitation speed of 220 R. P. M. for approximately 72 hours while maintaining the temperature at 28 C. A sterile solution of 20 mg. of 4- pregnene-3,20-dione in 3 m1. of propylene glycol was added to each flask and the agitation continued for approximately 48 hours. At this point 50 ml. of a culture growth, having initially the composition of medium #1 and containing 5 ml. of vegetative growth of Trichoa'erma nigrovirens, was added to each flask. The culture growth was obtained by developing the microorganism for three days in the medium. The flasks containing approximately 100 ml. were agitated at the same rate for 48 hours. One of the flasks was then extracted with chloroform and spotted on a paper strip. The spot was in the region of 4-pregnene-l7a,21-diol-3,20-dione. Fifty ml. of a culture growth having initially the composition of medium #1 and containing 5 m1. of vegetative growth of Stigmina platani, were added to each of the remaining flasks. The culture growth was obtained by developing the microorganism for three days in the medium. The flasks now containing approximately 150 ml. were again agitated for 24 hours and extracted with chloroform. The solutions were combined and evaporated to dryness and spotted and developed on paper strips. A tetrazolium reducing composition was found directly opposite 4-pregnene-l1p,i7iz, 21-triol-3,20-dione.

Any departure from the above description which conforms to the present invention is intended to be included within the scope of the claims.

What is claimed is:

1. A process for producing a polyhydroxy steroid of the group consisting of pregnenes, pregnanes, and allopregnanes having hydroxyl groups in at least two of the positions 11, 17a, and 21, which comprises subjecting a desoxy steroid of the group consisting of pregnenes, pregnanes, and allopregnanes, corresponding to the desired polyhydroxy steroid to the action of oxygenated strains of at least two diflerent types of microorganisms, one microorganism being active selectively oxygenating one of the positions 11, 17a, and 21, and a second microorganism being active in selectively oxygenating a different position of the group 11, 17a, and 21, in which the oxygenation by at least one of the microorganisms is carried out in the presence of at least one other microorganism to produce the corresponding polyhydroxy steroid.

2. The process according to claim 1, in which the conversions are carried out in the presence of growing cells of the respective microorganisms in a single nutrient medium and the product polyhydroxy steroid is separated from the microorganisms at the conclusion of the entire oxygenation process.

3. A process for producing a polyhydroxy steroid of the group consisting of pregnenes, pregnanes, and allopregnanes having hydroxyl groups in the 11, 17a, and 21 positions, which comprises subjecting a desoxy steroid of the group consisting of pregnenes, pregnanes, and allopregnanes, corresponding to the desired polyhydroxy steroid to the action of oxygenating strains of at least three different types of microorganisms, one microorganism being active in selectively oxygenating the 11- position of the steroid, 21 second microorganism being active in selectively oxygenating the 17t1-pOSllIiOl1, and a third microorganism being active in selectively oxygenating the 21-p0sition, in which oxygenation by at least one of the microorganisms is carried out in the presence of at least one other microorganism to produce the corresponding polyhydroxy steroid.

4. The process according to claim 3, in which one of the microorganisms is active in selectively introducing a hydroxy group at the llfi-position, and the product is a polyhydroxy steroid of the group consisting of pregnenes, pregnanes, and allopregnanes having hydroxy groups at the 115, 17a, and 21 positions.

References Cited in the file of this patent UNITED STATES PATENTS 2,602,769 Murray July 8, 1952 2,649,400 Murray et al Aug. 18, 1953 2,649,402 Murray et al Aug. 18, 1953 2,658,023 Shull et al Nov. 3, 1953 2,671,096 Murray ct a1. Mar. 4, 1954 OTHER REFERENCES Bessey: Morphology and Taxonomy of Fungi, The Blakiston Company, Phila., 1950, pp. 15-18. 

1. A PROCESS FOR PRODUCING A POLYHYDROXY STEROIS OF THE GROUP CONSISTING OF PREGNANES, AND ALLOPREGNANES HAVING HYDROXYL GROUPS IN AT LEAST TWO OF THE POSITIONS 11, 17A, AND 21, WHICH COMPRISES SUBJECTING A DESOXY STEROID OF THE GROUP CONSISTING OF PREGNANES, PREGNANES, AND ALLOPREGNANES, CORRESPONDING TO THE DESIRED POLYHYDROXY STEROID TO THE ACTION OF OXYGENATED STRAINS OF AT LEAST TWO DIFFERENT TYPES OF MICROORGANISMS, ONE MICROORGANISM BEING ACTIVE SELECTIVELY OXYGENATING ONE OF THE POSITIONS 11, 17A, AND 21, AND A SECOND MICROORGANISM BEING ACTIVE IN SELECTIVELY OXYGENATING A DIFFERENT POSITION OF THE GROUP 11, 17A, AND 21, IN WHICH THE OXYGENATING BY AT LEAST ONE OF THE MICROORGANISMS IS CARRIED OUT IN THE PRESENCE OF AT LEAST ONE OTHER MICROOGANISM TO PRODUCE THE CORRESPONDING POLYHYDROXY STEROID. 